Apoptosis is the orchestrated process by which cells self-destruct in response to specific internal and external signals. Programmed cell death is regulated by a complex network of checks and balances governed by the "BCL-2 family" of proteins. Faulty regulation of apoptotic pathways is a seminal event in the pathogenesis of a variety of hematologic and other diseases. BCL-2 is a survival protein, and its overproduction facilitates pathologic cell survival death. The specific molecular choreography that triggers mitochondrial apoptosis remains unknown. Bid's structure is defined by 8 alpha-helices, one of which is a critical death effector helix. We hypothesize that specific alpha-helical domains within BID engage in distinct interactions that contribute to its overall function. The goal of this research is to fuse chemical and biological techniques to elucidate the mechanism of BID-induced apoptosis and develop molecules to manipulate apoptosis in vivo. Thus, the specific aims of this proposal are: 1) Synthesize and characterize Stabilized Alpha Helices of BID (SAHBs), 2) Define the role(s) of BID's alpha-helical domains in triggering mitochondrial apoptosis, and 3) Utilize SAHBs to validate and manipulate BID activities in cultured cells and mouse models. A preliminary panel of SAHBs modeled after BID's death effector helix has been synthesized. Compared to unmodified peptide, two of these SAHBs demonstrate: (a) more than 5-fold increase in alpha-helicity; (b) up to 3.5-fold enhancement in protease resistance; (c) up to 25-fold increased potency in mitochondrial cytochrome c release, a key step in apoptosis; and (d) apoptosis induction in cultured Jurkat cells. SAHBs have the dual potential to serve as biological tools to dissect apoptotic pathways, and as prototype therapeutics for diseases characterized by aberrant cellular hypo- or hyperplasia. A diverse team of internationally recognized mentors, collaborators, and advisors with expertise in the fields of synthetic chemistry, structural biology, apoptosis biology, and clinical hematology/oncology will provide an idea training environment for Dr. Walensky's development as an independent investigator at the interface of these notable fields.